1. Field of the Invention
This invention relates to a method of purifying Factor VIII:C which is useful for therapeutic administration to patients having hemophilia.
Hemostatis, the biochemistry of blood coagulation, is an extremely complex and as yet not completely understood phenomena whereby normal whole blood and body tissue prevent an excess loss of blood from a ruptured blood vessel. The total mechanism of blood coagulation is affected through the coordinated interaction of biochemical substances contained in three basic physiologic systems; namely, extravascular tissue such as subcutaneous tissue, muscle tissue, and skin; the blood vessel wall; and intravascular components, including blood plasma proteins, blood plasma factors, and platelets.
A great deal of medical research into blood clotting diseases has been directed towards finding an acceptable treatment for hemophilia, a genetically induced disease characterized by the loss of clottability of otherwise normal whole blood. Although the precise cause of hemophilia is not known, one of the most popular theories suggests that it may be because of the absence of or a greatly inhibited presence of the active form of antihemophilic factor (hereinafter AHF) in otherwise normal plasma from whole blood. At present, although hemophilia cannot be cured, it can often be treated therapeutically by the administration of AHF to an AHF-deficient individual. The administered AHF is derived from blood obtained from a normal and healthy donor. AHF is administered either by the transfusion of whole blood or blood plasma, or by the infusion of AHF plasma protein concentrate which has been extracted from the plasma of normal human whole blood.
When whole blood or blood plasma transfusions are used to relieve a hemophiliac, one must exercise great care to select reasonably fresh blood or plasma because the biologic activity of AHF is extremely labile upon storage under normal conditions. Even laboratory techniques, such as lyophilization and cryogenic preservation, will not prevent substantial loss of biologic activity of AHF over time. Another major disadvantage of whole blood or blood plasma transfusions is that they can introduce unwanted proteinaceous and nonproteinaceous material in the recipient's blood stream, often causing allergic reactions to sensitive patients, viral infections such as hepatitis, or hypervolumetric reactions to those persons who require extensive amounts of AHF to initiate clotting.
Another method of therapeutic technique, namely, i.v. administration of AHF plasma concentrate, is presently being used extensively. These concentrates are being developed primarily to circumvent the aforementioned troublesome and often times dangerous side effects caused by whole blood or plasma transfusions.
Essentially, AHF plasma concentrate might be characterized as AHF-rich blood plasma extracts from which some blood plasma proteins, such as the gamma globulins, most other blood plasma factors, and many inorganic chemicals have been removed. However, even currently available AHF-rich blood plasma concentrates may contain impurities which can cause deleterious effects when administered to man so that a need for a purer, more therapeutically acceptable AHF plasma concentrate still exists.
AHF in its natural form as obtained from plasma consists of aggregates of two molecular entities, which are termed Factor VIII:R and Factor VIII:C. Factor VIII:C is biologically active in correcting the coagulation defect of Hemophilia A. Factor VIII:R, also known as Factor VIII:WF (von Willebrand Factor), is biologically active in correcting the coagulation defect of von Willebrand's disease, a disorder of platelet aggregation. During plasma fractionations designed to generate AHF rich extracts, Factor VIII:C and Factor VIII:R usually remain closely associated in a high molecular weight complex. Nevertheless, dissociation occurs in buffers containing high concentrations of calcium chloride, or sodium chloride at low pH values.
These conditions for dissociation are used in conjunction with chromatography of AHF plasma concentrates on aminohexyl-sepharose, quaternary aminoethyl-sepharose, or polyelectrolytes to separately elute Factor VIII:C and Factor VIII:R, often with reduced contamination by other plasma proteins. It is highly desirable to be able to purify Factor VIII:C with respect to Factor VIII:R and the other plasma proteins with which Factor VIII:C is normally found in order to provide antihemophilic therapy without risks of antigenic or viral side effects.
The present invention is directed to a method for obtaining highly purified Factor VIII:C having high specific activity for initiating clot formation intended to be used therapeutically to correct a clinical defect known as severe hemophilia A.
2. Description of the Prior Art
Various protein concentrates are known to contain Factor VIII:C activity, including human and animal plasma, such as described in U.S. Pat. No. 4,210,580 and extracts from cell cultures which have been genetically engineered to contain Factor VIII:C, such as described by Wood, et al, in Nature, Vol. 312, pp 330-336 (1984). Protein concentrates containing Factor VIII:C activity can be produced from the material referred to hereinabove by a variety of methods. Crude Factor VIII:C concentrates typically have potencies of 10 to 20 units per ml and purities of 1 to 5 units per mg.
Previously known processes for purifying Factor VIII:C introduce losses of yield and/or purity which up to now have been tolerated. The present invention achieves higher levels of purification and yield, without deactivation, in a manner which is not suggested by the prior art.
D. E. G. Austen, "The Chromatographic Separation of Factor VIII on Aminohexyl Sepharose", in British Journal of Hematology, 1979, (43) 669-674, described a chromatographic separation process in which human or porcine Factor VIII concentrate was passed through a column of 6-amino-n-hexyl-substituted agarose. The column and all eluting solutions were at a pH of 5.5. A high degree of separation of Factor VIII:C from Factor VIII:R, and a high degree of purification of Factor VIII:C from other proteins, were obtained. However, the total recovery of human Factor VIII:C was only 35-40%, and for porcine Factor VIII:C was only 24-30%. The authors indicate that more acidic pH values in the buffers (down to a pH of about 5.2) favor higher purification of Factor VIII:C, and they purposely chose the pH of 5.5 in order to have as acidic an environment as possible without suffering too low a yield.
Several recent publications have continued to insist on maintaining an acid pH in the chromatographic column. Morgenthaler, "Chromatography of Antihemophilic Factor on Diaminoalkane- and Aminoalkane-Derivatized Sepharose", Thromb. Haemostas. 47(2) 124-127 (1982), found that when AHF was chromatographed on Sepharose CL-2B agarose gel at pH values of 6.0, 6.5 and 7.0, no significant separation of Factors VIII:C and VIII:R could be obtained. Chromatography of AHF at a pH of 5.5 produced a very marked separation between Factors VIII:C and VIII:R. An even more recent paper, Faure, et al., "Improved buffer for the chromatographic separation of Factor VIII coagulent," J. Chromatography 257 (1983), 387-391, retains the pH value of 5.5 indicated by Austen and attempts to improve the performance of that chromatographic procedure by adding compounds to the buffers.
Factor VIII:C has been purified from crude AHF using a column at a pH closer to neutral only in the instance in which Factor VIII:R is employed to form a reversible complex with Factor VIII:C. The Factor VIII:C is then eluted in the presence of a neutral aqueous buffer containing at least 0.2M calcium chloride. Specifically, in U.S. Pat. No. 4,361,509, Zimmerman, et al. employ a column bearing monoclonal antibodies to Factor VIII:R to recover a dilute solution of Factor VIII:C that has been ultrapurified free of most Factor VIII:R.
The solution of ultrapurified Factor VIII:C obtained from either of the above techniques can be concentrated at a neutral pH around 6.8 using selected columns of diaminoalkane or aminoalkane derivatized agarose.
For diaminoalkane agarose, such as aminohexyl agarose, the salt concentration of the ultrapure Factor VIII:C solution must first be lowered to around 0.05M for efficient binding. The factor VIII:C is then eluted from the column with a buffer containing high concentrations of calcium chloride or sodium chloride at a neutral pH.
One type of aminoalkane derivatized agarose, butyl agarose, has also been used to concentrate ultrapurified Factor VIII:C solution following a similar manipulation of salt concentrations.
Other types of aminoalkane derivatized agarose such as pentyl-, hexyl-, heptyl-, and octyl-agarose as well as phenyl-agarose are generally known as hydrophobic interaction matrix.
Ultrapurified Factor VIII:C has been shown in the prior art to bind to each of the hydrophobic interaction matrix, however no active material has been recovered following elution with the salt solutions typically used by those skilled in the art. Recovery to a large degree is determined by the affinity of AHF to the hydrophobic interaction matrix from which AHF is difficult to separate by the use of salt solutions with various salt concentrations.
We have now discovered that the use of certain surface active agents in the elution buffer results in an effective recovery of AHF activity from hydrophobic interaction matrix.